Many protein products are freeze-dried to provide adequate shelf life stability. M. J. Pikal, K. M. Dellerman, M. L. Roy and R. M. Riggin, Pharmaceutical Research 1990, 8(4), 427-436. To be successful, a freeze-dried product survive processing and storage over the claimed shell life without excessive loss of potency or excessive increase in the level of decomposition products. Human growth hormone (hGH) is an example of a protein that may be freeze-dried without significant degradation, but the resulting solid is potentially unstable. M.J. Pikal, K. M. Dellerman and M. L. Roy, Develop. biol. Standard, 1991, 74, 21-38.
Protein stability is more complex in heterogeneous systems such as protein formulations, with the extent of the stability problem being sensitive to both formulation variables and the level of residual water in the freeze-dried solid. Water content can vary depending on the freeze-drying process, and may increase during storage. M.J. Pikal and S. Shah, Develop. biol. Standard, 1991, 74, 165-179. Moreover, low residual moisture after manufacture does not ensure low moisture content throughout the shelf life of the product. Freeze-drying of pharmaceuticals for parenteral use is generally performed in the final container, such as a glass ampule or vial. The rubber stoppers typically used to seal the final containers contain a measurable quantity of water, which can transfer to the freeze-dried product until the water content in the product eventually reaches equilibrium with the water content in the stopper.
A low residual moisture content in a freeze-dried protein product is essential to maintain the stability of compounds that are prone to hydrolysis. High water content may decrease protein stability via several mechanisms. The effect of water content and/or water activity on the solid state stability of proteins results from either changes in dynamic activity or conformational stability of the protein, or participation of water as a reactant or medium for mobilization of reactants. Chemical modification generally results in changes to the primary sequence and may or may not have a subsequent effect on conformational structure. Water is a reactant in the deamidation reaction, and high levels of water should increase the rate of deamidation. M. J. Hageman, Drug Dev. Ind. Pharm,, 1988, 14, 2047-2070. Residual water in excess of monolayer coverage also increases molecular mobility in the solid protein, thereby increasing general reactivity.
Due to the key role water content plays in the solid state of protein formulations, an accurate and precise moisture determination method is essential in resolving stability issues and in setting specifications and criteria for acceptable moisture content. A number of chromatographic, spectroscopic, electronic, thermal, and wet chemical methods have been used in the past to determine moisture levels. S. K. MacLeod, Anal. Chem., 1991, 63, 557A-565A. The most common of these are loss on drying (LOD), thermogravimetric analysis (TGA), gas chromatography using a thermal conductivity detector, and the Karl Fischer titration.
Of these most common water content measurements, however, the Karl Fischer titration has become the method of choice and is now the approach most widely used in the determination of water content. The direct titration of, or assay for, water using KF reagent is currently in all of the official compendia, and is implied in several official methods manuals (i.e., USP, EP). The titration can be run in either protic or aprotic medium, with the protic medium seeing wider use due to higher sensitivity of the titer to sample and solvent composition. M. S. Kamat, R. A. Lodder and P. P. DeLuca, Pharmaceutical Research, 1989 6(11) 961-965. The reaction in protic media (i.e., alcohol) involves sulfur dioxide reacting with the alcohol to produce an alkyl sulfite in a buffered medium using an appropriate base to maintain the solution at the optimal pH. In a coulometric experiment, the iodine is generated electrically from iodine present in the cell The electric efficiency of this method is generally 100%, and the amount of water in the sample is calculated from the number of moles of electrons used in the iodine generation.
An important point to consider with the Karl Fischer titration is the possibility of erroneous results due to water contamination. Titration of small quantities of water in coulometric systems requires a correction for atmospheric moisture entering the system. Sample handling can have a significant impact on the results of a titration as a result of the gain or loss of moisture between sampling and analysis. This leads, at times, to erroneous conclusions because, for example, many lyophilized samples with extreme hygroscopicity pick up atmospheric moisture during sample handling. This becomes increasingly frustrating when one considers the increased variability of the assay due to wide fluctuations in relative humidity throughout the year. Precision in the method is governed largely by the extent to which atmospheric moisture is excluded from the system and the sample.
Accurate moisture content determination measurements using the Karl Fischer titration is contingent on the proper working order of the titration instrument and the chemical reactions. Successful moisture content determinations require 1) that equipment be in proper working order, 2) that reagents be stable and not depleted, 3) that moisture be excluded from the system, 4) that the anodic reaction produce 100% current yield, 5) that the cathodic reaction does not interfere with the titration, and 6) that the reaction not be adversely affected by the sample matrix. To assure that these criteria are being met, the quality of the analysis is checked against standards containing a known moisture content. The correct moisture content determination for the standards confirms that the Karl Fischer titration analysis is running properly, or indicates that a problem exists. A variety of materials have been proposed as standards for moisture content determinations. The principal requirements of these materials are 1) that they contain a stoichiometric amount of moisture that is stable over a wide range of temperature and humidity, 2) solubility in the Karl Fischer titration reagents, 3) ease of handling and storage, 4) availability, and 5 ) uniformity. M. S. Kamat, R. A. Lodder and P. P. DeLuca, Pharmaceutical Research, 1989, 6(11), 961-965.
Much effort has been given to making liquid water standard solutions less hygroscopic. These efforts have not been completely successful as the water content of the solutions change after the septum over the solutions has been pierced several times. Water is a very good calibration reagent, but it is difficult to accurately dispense liquid water into the Karl Fischer titrator. When delivered by volume, the inaccuracies of the small amount delivered makes it difficult to obtain an accurate value. A more accurate measurement is obtained when the liquid water is delivered by weight, but this again presents difficulties in dispensing the water into the titrator.
Solid water standards have many useful characteristics, but solids such as sodium tartrate dihydrate are not easily dissolved in many Karl Fischer titration reagents, E. Scholz, Karl Fischer Titration-Determination of Water-Chemical Laboratory PracticeSpringer-Verlag, N.Y. 1984, and questions regarding uniformity of supplies have been raised. T. H. Beasley, H. W. Siegler, R. L. Charles and P. King, Anal. Chem., 1972, 44, 1833-1840. It has been observed that the moisture content value of the Hydrahal.RTM. solid standard (sodium tartrate dihydrate) varied widely depending from where the sample is taken within the sample container. Sampling from the top of the bulk material (skimming the top) resulted in moisture content values approximately 15% less than the expected value and 10% less than the average value of samples taken deeper in the container. The UpJohn Company has found that a lincomycin hydrochloride monohydrate standard does not change water content over a wide range of relative humidities, and is quite stable. The availability of this material, however, is unclear because it is a prescription pharmaceutical. M. S. Kamat, R. A. Lodder and P. P. DeLuca, Pharmaceutical Research, 1989, 6(11), 961-965.
Due to the foregoing concerns regarding sample handling and accurate water standard determinations, the usefulness of coulometric moisture determinations as currently performed in measuring residual moisture is questionable. This is no better demonstrated than in the analysis of formulations of human growth hormone in vials and cartridges. The stability of this product is adversely affected by high moisture content in the lyophilized plug and is quickly affected by environmental moisture contamination upon exposure to the laboratory atmosphere. In the past, the vialed material was weighed on a balance, the rubber stopper removed, the lyophilized plug broken up into a powder with a spatula, the contents dispensed into the titrator using a glass funnel, the rubber stopper placed back on the vial, and the vial reweighed on the balance. The difference in weight was punched into the Karl Fischer titrator to be used for the percent moisture content value.
The time to transfer the material, from the time of removing the rubber stopper to the time of dispensing the entire contents of the vial, depended on how adept the operator was in performing the transfer. The faster the transfer, the less exposure to the lab atmosphere, and the less moisture content contamination. This is well illustrated in FIG. 6, which shows the dramatic effect of transfer time on moisture content values for a 5 mg formulated vial of hGH at a laboratory relative humidity level of 40%. The fastest one of the inventors, with several months experience, was able to transfer the contents of the vial into the reaction vessel in 28 seconds. Transfer times greater than this minimum transfer time resulted in a sharp increase in moisture content, with a doubling of the transfer time resulting in a 125% increase in moisture content for this material. Transfer times greater than approximately one minute resulted in little increase in moisture content, indicating an equilibrium with the laboratory atmosphere.
This data raised the question of the extent of moisture contamination prior to the 28 second minimum transfer time. One can assume that moisture contamination is occurring as soon as the rubber stopper is removed from the vial. This measured moisture content value therefore has little correlation to the actual moisture content in a closed vial due to the physical limitations of this material transfer process. This prior art process is confounded further by the fact that the relative humidity in a lab typically varies from day to day and from month to month. The relative humidity in the inventors' laboratory has varied from 15% to 50% during a one month period.
Another problem with the sample transfer process of the prior art is dispensing the lyophilized material into the Karl Fischer titrator. The material is rather flaky, and when trying to pour the material through the funnel, some material is lost into the atmosphere. The material also adheres to the sampling funnel and is not all dispensed into the titrator. To get around this, weighing paper can be rolled to crate a funnel, but this takes some operator dexterity, during which the titrator is open to the atmosphere. In its prior art configuration, Karl Fischer titrations of hGH formulations were affected by: 1) sample transfer time, 2) relative humidity in the laboratory, and 3) material lost in the material transfer. These factors make it impossible to determine the accurate moisture content in the closed vial.